The binding of sickle hemoglobin to the cytoplasmic domain of protein 3 is emerging as a central interaction in the pathogenesis of membrane damage in sickle cell disease. This interaction targets toxic iron species to vulnerable membrane proteins and ultimately causes protein 3 to cluster, a rearrangement that also involves ankyrin and glycophorin. At the outer surface of the cell, the clustered proteins are recognized by autoantibodies in the plasma and become decorated with immunoglobulin and complement. This signal incites macrophages to remove the cells prematurely from circulation. Additional potential effects of this interaction include clustering of other membrane proteins, modulation of cellular metabolism, and alteration of important structural membrane protein-protein interactions. Our hypothesis is that the composition of the cytoplasmic domain of protein 3 is an important variable in this pivotal interaction, and has an impact on the pathophysiology of sickle cell disease. We will use three experimental systems to study the influences of a variety of cytoplasmic domain structures. 1) We will use a system we design to test protein-3 hemoglobin interactions by measuring the influence of cytoplasmic domain of protein 3 on the hemoglobin dimer- tetramer equilibrium. We will test purified native cytoplasmic domain of protein 3 as well as specifically-designed mutant peptides to map the key residues on both the hemoglobin and protein 3 side of the interaction. 2) We have found an unexpectedly high prevalence of a cytoplasmic domain of protein 3 mutant (Memphis, 56-->GLU) in sickle cell anemia and hypothesize that its presence increases disease severity. We will define the impact of this mutation on biochemistry, physiology, hematology, and clinical severity. 3) The cytoplasmic domain of protein 3 is poorly conserved in mice, and we hypothesize that its interaction with human sickle hemoglobin will be reduced, limiting the comparability of the mouse model. We will compare the physiologic, biochemical, and clinical phenotype to the conventional mouse model.